Cell growth for extract preparation was performed using BL21 Star (DE3) cells harboring the pEVOL-pPrF plasmid (39) in 2.5-L baffled tunair flasks (IBI Scientific, Peosta, IA, USA). Cells were cultured at 37°C with 280 rpm agitation in a rotary incubator shaker (value in g not specified for all incidences of rpm). The fermentation was performed with and without the presence of 100 mM 3-morpholinopropanesulfonic acid (MOPS) in 2× yeast extract and tryptone (YT) growth media. The fermentations were inoculated with 1 mM isopropyl β-D-1-thiogalactopyranoside and 0.02% (w/v) L-arabinose at an optical density (OD600) of 0.6. Cells were harvested at mid to late logarithmic growth phase at an OD600 of 2.7 to 3.8 4 h after induction by centrifugation at 8000 rpm at 4°C for 30 min. Cells were then washed by suspending in 10 mL ice-cold buffer A (10 mM Tris base, 14 mM magnesium acetate, 60 mM potassium glutamate, and 1 mM dithiothreitol) per gram of cell and centrifuged at 6000 rpm at 4°C for 30 min and subsequently resuspended in 1 mL ice-cold buffer A per gram of cell in preparation for cell lysis. Finally, the cell suspension was flash-frozen in liquid nitrogen and stored at -80°C prior to lysis. Note that the pEVOL-pPrF plasmid and its induction with arabinose are not necessary for extract preparation. However, its inclusion in the E. coli cells used for extract preparation enables the option of cell-free production of proteins containing unnatural amino acids.

Cell lysis and extract preparation

High-pressure homogenization. Thawed cell suspensions were lysed with three passes through an Emulsiflex-B15 French press-style high-pressure homogenizer (Avestin, Ottawa, ON, Canada) at 24,000 psi with sample cooling for 1 min in an ice-water bath after the second pass. The lysate was centrifuged at 12,000× g for 10 min at 4°C, and the pellet was discarded. The supernatant was carried forward for a run-off reaction by incubating at 37°C with 280 rpm agitation for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use.

Sonication. Thawed cell suspensions were lysed using a Vibra-cell VCX 400 probe sonicator with a CV 26 probe (tip diameter of 3 mm; Sonics & Materials, Newtown, CT, USA) at a frequency of 20 kHz. The sample vial was kept in an ice-water bath to prevent significant heating in the sample during sonication. The lysate was centrifuged for 30 min at 12,000× g at 4°C, and the run-off reaction was performed by incubating the supernatant at 37°C with 280 rpm agitation for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use. Sonication was performed by cycling at the sonication and cooling intervals as shown in Table 1.

Bead vortex mixing. Glass beads of 0.1 mm diameter (Scientific Industries, Bohemia, NY, USA) at 10%, 20%, 50% or 80% (w/v) beads-to-cell suspension ratio were used for cell lysis in 1.5- or 2-mL microcentrifuge tubes. The cell and bead suspension was vortex mixed on the table top vortex mixer, Fisher Vortex Genie 2 (Scientific Industries), at 3200 rpm for different time intervals with a 1-min cooling period between each vortex mixing. The lysate was centrifuged twice at 12,000× g at 4°C for 30 min with the supernatant retained each time. The run-off reaction was performed at 37°C and 280 rpm shaking for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use.

Lysozyme incubation. Hen egg white lysozyme (Sigma-Aldrich, St. Louis, MO, USA) was added to the thawed cell suspension at a concentration of 0.1 mg/mL and incubated at 37°C with gentle shaking (80 rpm) for 3 h, similar to as reported previously (40). The lysate was centrifuged at 12,000× g at 4°C for 10 min. The run-off reaction was performed by incubating the supernatant at 37°C and 280 rpm shaking for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use.

Freeze-thaw cycling. Thawed cell suspensions of 500 µL in 1.5-mL microcentrifuge tubes were subjected to three freeze-thaw cycles by freezing the cell suspensions on liquid nitrogen or dry ice for 15 min and thawing for 15 min in a water bath at 25°C, similar to previously reported methods (41, 42). The lysate was centrifuged at 12,000× g at 4°C for 10 min. The run-off reaction was performed by incubating the supernatant at 37°C and 280 rpm shaking for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use.